5 Techniques in Protein Analysis

• Mechanical disruption is a method that physically breaks down the cell or the tissue to release the proteins.

  • It can be achieved thru blending using blender
• Liquid homogenization is a physical method where the sample is mixed with liquid medium to create a homogenous mixture.

  • It facilitates the release of the proteins by disrupting the cellular structures.
• High-frequency sound waves or Sonication is a physical method that utilizes high frequency sound waves to disrupt the cells and release the proteins.

  • Sound waves will generate alternating cycle of compression and refraction causing the formation of collapse of small bubbles.
  • The resulting shock waves will disrupt the cellular structures and it will in protein extraction
• Freezing or Thaw cycles is a physical method that exploit the freezing and thawing process to disrupt the cell enabling the protein release
• Manual Grinding is a physical method that is done through the use of mortar and pestle
• Detergent-based is a chemical method that employs the use of detergents to solubilize the proteins and disrupt the cellular membrane.
• Detergent can dissolve lipid bilayers that will facilitate the release of the membrane associated proteins
• Enzymatic methods involves the use of specific enzymes to degrade cellular components and facilitates proteins isolation.
• Enzymatic methods

  • Nucleases, RNase, DNase can be used to degrade nucleic acids
  • Lysozyme can digest the polysaccharide components of the cell making it easier to lyse
• Column Chromatography is widely used technique for protein separation

  • Involves passing a protein mixture through a column pack with a stationary phase (often a porous solid matrix), and a mobile phase, which is a liquid solvent
  • Proteins will interact differently with the stationary phase, which is based on their properties leading to separation
• Column chromatography example:

  • Proteins with higher affinity for stationary phase will move more slowly through the column while proteins with lower affinity will elude faster.
  • The choice of stationary phase depends on the desired separation mechanism
• Ion-Exchange Chromatography separates proteins based on their charge differences (based on the word itself “ion” meaning there’s a positive and negative charge)

  • Stationary phase in this technique contains charged groups. It is either positive or negatively charged
  • Proteins with opposite charge to the stationary phase bind to it, while proteins with similar charges will pass through more quickly.
  • By adjusting the pH or the salt concentration of the mobile phase bound proteins can be eluted selectively based on their charge.
• Gel-Filtration Chromatography AKA Size Exclusion Chromatography separates proteins based on their size or molecular weight

  • The stationary phase consist of a porous gel matrix with define 4 sizes
  • Smaller proteins penetrates the pores and take longer to pass through the column, while larger proteins move more rapidly
  • Consequently, proteins are eluted in order of decreasing molecular weight allowing for their separation based on their size
• Affinity Chromatography exploits the specific interaction between protein of your interest and a ligand or affinity resin

  • The stationary phase is typically a matrix to which a ligand is attached.
  • Protein of interest binds selectively to the ligand allowing to be retained on the column while other proteins flow through
  • The bound protein is subsequently eluted using as specific elution buffer or by altering the conditions of the column to disrupt the protein ligand interaction
• SDS-PAGE employs Polyacrylamide gel and anion detergent called Sodium Dodecyl Sulfate to denature proteins and facilitate their separation
• • Electrophoresis → a method that exploit the movement of charge molecules.
  • Gel Electrophoresis → a form of zone electrophoresis utilizes a gel matrix as a molecular sieve to separate molecules base on their size
• SDS-PAGE

  • Since all proteins have a net negative charge, proteins are repelled by negative cations and attract to the positive anode.
  • As proteins move toward the anode, polyacrylamide measured obstructs and slows larger proteins, but allows smaller proteins to move faster
  • In consequence, the distance traveled in a given time is proportional to the log of the molecular weight. After separation, the proteins are visualized using a dye.
• The structure of the SDS is amphipathic that is long hydrocarbon tail, and the sulfate is hydrophilic part.
• In SDS-PAGE, the first step to separate a mixture of proteins by size is to boil the sample.
• Boiling proteins in a solution of SDS will destroy the tertiary structure of the proteins

  • RESULT: unfolded protein with negative charge that is proportional to its molecular weight
• Vertical electrophoresis system is utilized in SDS-PAGE
• In Continuous buffer system, gel and tank buffers are the same

  • The buffer maintains a constant pH and ionic strength
  • Stable and uniform electrophoretic environment
• In Discontinuous buffer system, gel and tank buffers are the different.

  • Gel is divided into stacking and running gel
• Stacking gel has lower acrylamide concentration

  • weakly buffered at pH 9.0
  • lower ionic strength (high electrical resistance)
• Running gel has higher acrylamide concentration

  • strongly buffered at pH 9.0
  • high ionic strength (lower electrical resistance)
• Western Blot is used to detect specific protein, it could be antigen or antibody.

  • We use probes here, which can also be antigen or antibody, depending on what kinds of proteins are you detecting
• Bradford method or Coomassie protein assay

  • Developed by Marion M. Bradford
  • Based on the equilibrium between 3 forms of Coomassie Blue G dye
• Bradford method is under strongly acidic conditions.

  • The dye is most stable as doubly-protonated red form
  • Upon binding to protein, it is converted to a stable unprotonated blue form
• Bicinchoninic Acid Assay

  • Developed by Paul K. Smith
  • Copper-based colorimetric assay for total protein quantification
  • Observed and measure at 562 nm (550-570 nm)
• Principle of BCA assay is the reduction of the cupric ions to cuprous ions by protein in an alkaline medium followed by a formation of colored complex with the BCA

  • BCA reacts with cuprous ions forming purple colored complex, which is the end product
• Enzyme-linked Immunosorbent Assay

  • First described by Eva Engvall and Peter Perlmann in 1971
  • A widely used laboratory technique for detecting and quantifying specific proteins, antibodies, antigens, or other molecules.
  • Utilizes enzyme-mediated colorimetric or fluorescent detection
• The principle of ELISA is antigen-antibody binding
• In Direct ELISA, the target antigen is immobilized onto a solid surface such as microplate

  • Directly detected using labeled primary antibody, which is specific to your target antigen Primary antibody binds directly to the antigen, and the labeled attached to the primary antibody provides a detectable signal
  • This method is relatively simple and quick, but it may have limitations in terms of sensitivity and availability of suitable primary antibodies
• In Indirect ELISA, the target antigen is immobilized onto a solid surface, and it is detected using two antibodies

  • This method offers increase sensitivity compared to direct ELISA, as multiple secondary antibodies can bind to a primary antibody which enhancing the signal
• Antibodies used in Indirect ELISA

  • Primary antibody – specific to target antigen and it binds to it
  • Secondary antibody - labeled w/ Enzyme → gives signal; recognizes and bind to the primary antibody and it will give us the signal
• In Sandwich ELISA, the target antigen is captured between two antibodies

  • It is referred to as captured antibody and the detection antibody
• Antibodies used in Sandwich ELISA

  • Captured antibody – immobilized onto the solid surface and it binds to the antigen present in your sample
  • Detection antibody – labeled with an enzyme or a fluorophore is added; recognizes and binds to different epitopes of antigen forming a “sandwich complex”
• In Competitive ELISA, the target antigen competes with a labeled antigen for binding into a limited amount of specific antibodies.

  • The solid surface is typically coated with captured antibodies or antigens, which will recognize and binds to your target antigen or antibody
  • The labeled antigen is added to the sample along with the antibody/antigen solution

  The signal is inversely proportional to the concentration of the target antigen
• Positive sample in competitive ELISA has no color change, while negative sample, there is color change
• Mass spectrophotometry for protein identification is specific and sensitive technique for protein identification and characterization